Anti-inflammatory indole derivatives

ABSTRACT

Therapeutic compounds of formula (I) wherein X is CH 2  or SO 2 ; R 1  is an optionally substituted aryl or heteroaryl ring; R 2  and R 3  are various specified groups, R 4  is a group NHCOR 15 , NHSO 2 R 15  or OCONR 16 R 17  where R 15  is optionally substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl and R 16  and R 17  are independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl, with the proviso that at least one of R 16  or R 17  is other than hydrogen, or R 16  and R 17  together with the nitrogen atom to which they are attached from an optionally substituted heterocyclic ring which optionally contains further heteroatoms; and R 5 , R 6  and R 7  are independently selected from hydrogen, a functional group or an optionally substituted hydrocarbyl groups or optionally substituted heterocyclic groups; and further provided that when R 4  is a group NHCOR 15 , R 15  is substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl; as well as pharmaceutical compositions containing them are described and claimed. These compounds and compositions are useful in the treatment of disease mediated by monocyte chemoattractant protein-1 or RANTES (Regulated Upon Activation, Normal T-cell Expressed and Secreted), such as inflammatory disease.

The present invention relates to chemical compounds, to their productionas well as to pharmaceutical compositions containing them as well as totheir use in therapy, in particular of inflammatory disease.

MCP-1 is a member of the chemokine family of pro-inflammatory cytokineswhich mediate leukocyte chemotaxis and activation. MCP-1 is a C—Cchemokine which is one of the most potent and selective T-cell andmonocyte chemoattractant and activating agents known. MCP-1 has beenimplicated in the pathophysiology of a large number of inflammatorydiseases including rheumatoid arthritis, glomerular nephritides, lungfibrosis, restenosis (International Patent Application WO 94/09128),alveolitis (Jones et al., 1992, J. Immunol., 149, 2147) and asthma.Other disease areas where MCP-1 is thought to play a part in theirpathology are atherosclerosis (e.g. Koch et al., 1992, J. Clin. Invest.,90, 772-779), psoriasis (Deleuran et al., 1996, J. DermatologicalScience, 13, 228-236), delayed-type hypersensitivity reactions of theskin, inflammatory bowel disease (Grimm et al., 1996, J. LeukocyleBiol., 59, 804-812), multiple sclerosis and brain trauma (Berman et al,1996, J. Immunol., 156, 3017-3023). An MCP-1 inhibitor may also beuseful to treat stroke, reperfusion injury, ischemia, myocardialinfarction and transplant rejection.

MCP-1 acts through the MCP-1 receptor (also known as the CCR2 receptor).MCP-2 and MCP-3 may also act, at least in part, through the MCP-1receptor. Therefore in this specification, when reference is made to“inhibition or antagonism of MCP-1” or “MCP-1 mediated effects” thisincludes inhibition or antagonism of MCP-2 and/or MCP-3 mediated effectswhen MCP-2 and/or MCP-3 are acting through the MCP-1 receptor.

Copending International Patent Application Nos. PCT/GB98/02340 andPCT/GB98/02341 describe and claim groups of compounds based upon theindole ring structure which arc inhibitors of MCP-1 and therefore haveapplications in therapy.

The use of certain indole derivatives as NMDA antagonists is describedis U.S. Pat. No. 5,051,442, WO93 12780, EP-483881. Other indoles andtheir use as inhibitors of leukotriene biosynthesis is described in forexample, EP-A-275-667.

The applicants have found a particular substitution on the indole ringproduces advantageous results when used therapeutically as inhibitors ofMCP-1.

According to the present invention there is provided a compound offormula (I)

X is CH₂ or SO₂

-   R¹ is an optionally substituted aryl or heteroaryl ring;-   R² is carboxy, cyano, —C(O)CH₂OH, —CONHR⁸, —SO₂NHR⁹, tetrazol-5-yl,    SO₃H, or a group of formula (VI)    where R⁸ is selected from hydrogen, alkyl, aryl, cyano, hydroxy,    —SO₂R¹² where R¹² is alkyl, aryl, heteroaryl, or haloalkyl, or R⁸is    a group —(CHR¹³)_(r)—COOH where r is an integer of 1-3 and each R¹³    group is independently selected from hydrogen or alkyl; R⁹ is    hydrogen, alkyl, optionally substituted aryl such as optionally    substituted phenyl or optionally subtituted heteroaryl such as 5 or    6 membered heteroaryl groups, or a group COR¹⁴ where R¹³ is alkyl,    aryl, heteroaryl or haloalkyl; R¹⁰ and R¹¹ are independently    selected from hydrogen or alkyl, particularly C₁₋₄ alkyl;-   R³ is hydrogen, a functional group, optionally substituted alkyl,    optionally substituted alkenyl, optionally substituted alkynyl,    optionally substituted aryl, optionally substituted heterocyclyl,    optionally substituted alkoxy, optionally substituted aralkyl,    optionally substituted aralkyloxy, optionally substituted    cycloalkyl;

R⁴ is a group NHCOR¹⁵, NHSO₂R¹⁵ or OCONR¹⁶R¹⁷ where R¹⁵ is optionallysubstituted alkyl, optionally substituted aryl or optionally substitutedheteroaryl and R¹⁶ and R¹⁷ are independently selected from hydrogen,optionally substituted alkyl, optionally substituted aryl and optionallysubstituted heteroaryl, with the proviso that at least one of R¹⁶ or R¹⁷is other than hydrogen, or R¹⁶ and R¹⁷ together with the nitrogen atomto which they are attached form an optionally substituted heterocyclicring which optionally contains further heteroatoms; and

R⁵, R⁶ and R⁷ are independently selected from hydrogen, a functionalgroup or an optionally substituted hydrocarbyl groups or optionallysubstituted heterocyclic groups.

Suitably, where R⁴ is a group NHCOR¹⁵, R¹⁵ is substituted alkyl,optionally substituted aryl or optionally substituted heteroaryl.

Compounds of formula (I) are inhibitors of monocyte chemoattractantprotein-1. In addition, they appear to inhibit RANTES (Regulated uponActivation, Normal Tcell Expressed and Secreted), induced chemotaxis.RANTES is another chemokine from the same family as MCP-1, with asimilar biological profile, but acting though the CCR1 receptor. As aresult, these compounds can be used to treat disease mediated by theseagents, in particular inflammatory disease.

In this specification the term ‘alkyl’ when used either alone or as asuffix includes straight chained, branched structures. These groups maycontain up to 10, preferably up to 6 and more preferably up to 4 carbonatoms. Similarly the terms “alkenyl” and “alkynyl” refer to unsaturatedstraight or branched structures containing for example from 2 to 10,preferably from 2 to 6 carbon atoms. Cyclic moieties such as cycloalkyl,cycloalkenyl and cycloalkynyl are similar in nature but have at least 3carbon atoms. Terms such as “alkoxy” comprise alkyl groups as isunderstood in the art.

The term “halo” includes fluoro, chloro, bromo and iodo. References toaryl groups include aromatic carbocylic groups such as phenyl andnaphthyl. The term “heterocyclyl” includes aromatic or non-aromaticrings, for example containing from 4 to 20, suitably from 5 to 8 ringatoms, at least one of which is a heteroatom such as oxygen, sulphur ornitrogen. Examples of such groups include furyl, thienyl, pyrrolyl,pyrrolidinyl, imidazolyl, triazolyl, thiazolyl, tetrazolyl, oxazolyl,isoxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzothiazolyl,benzoxazolyl, benzothienyl or benzofuryl.

“Heteroaryl” refers to those groups described above which have anaromatic character. The term “aralkyl” refers to aryl substituted alkylgroups such as benzyl.

Other expressions used in the specification include “hydrocarbyl” whichrefers to any structure comprising carbon and hydrogen atoms. Forexample, these may be alkyl, alkenyl, alkynyl, aryl, heterocyclyl,alkoxy, aralkyl, cycloalkyl, cycloalkenyl or cycloalkynyl.

The term “functional group” refers to reactive substituents. They maycomprise electron-donating or electron-withdrawing. Examples of suchgroups include halo, cyano, nitro, C(O)_(n)R¹⁸, OR¹⁸, S(O)_(n)R¹⁸,NR¹⁹R²⁰, C(O)NR¹⁹R²⁰, OC(O)NR¹⁹R²⁰, —NR¹⁹C(O)_(n)R¹⁸, —NR¹⁸CONR¹⁹R²⁰,—N═CR¹⁹R²⁰, S(O)_(m)NR¹⁹R²⁰ or —NR¹⁹S(O)_(m)R¹⁸ where R¹⁸, R¹⁹ and R²⁰are independently selected from hydrogen or optionally substitutedhydrocarbyl, or R¹⁹ and R²⁰ together form an optionally substituted ringwhich optionally contains further heteroatoms such as S(O)_(m), oxygenand nitrogen, n is an integer of 1 or 2, m is 1 or 2.

Suitable optional substituents for hydrocarbyl groups R¹⁸, R¹⁹ and R²⁰include halo, perhaloalkyl such as trifluoromethyl, mercapto, hydroxy,carboxy, alkoxy, heteroaryl, heteroaryloxy, alkenyloxy, alkynyloxy,alkoxyalkoxy, aryloxy (where the aryl group may be substituted by halo,nitro, or hydroxy), cyano, nitro, amino, mono- or di-alkyl amino,oximino or S(O)_(n)R^(x) where n is as defined above and R^(x) is alkylsuch as C₁₋₄ alkyl.

Suitable substituents for these hydrocarbyl or heterocylic groupsinclude those listed above for R¹⁸, R¹⁹ and R²⁰.

Suitably R¹ is an optionally substituted phenyl, pyridyl, naphthyl,furyl or thienyl ring, and in particular is a substituted phenyl orpyridyl ring.

Suitable optional substitutents for R¹ in formula (I) include alkyl,alkenyl, alkynyl, halo, haloalkyl including perhaloalkyl such astrifluoromethyl, mercapto, alkoxy, haloalkoxy, alkenyloxy, alkynyloxy,hydroxyalkoxy, alkoxyalkoxy, alkanoyl, alkanoyloxy, cyano, nitro, amino,mono- or di-alkyl amino, oximino, sulphonamido, carbamoyl, mono ordialkylcarbamoyl or S(O)_(m)R²¹ where m is as defined above and R²¹ ishydrocarbyl.

Particular examples of substituents R⁵, R⁶ and R⁷, and where appropriatealso R⁴ include hydrogen, hydroxy, halo, optionally substituted alkylsuch as aralkyl, carboxyalkyl or the amide derivative thereof; alkoxy;aryloxy; aralkyloxy; or an amino group which is optionally substitutedwith alkyl, aryl or aralkyl. A specific functional group which issuitable for R⁴, R⁵, R⁶ and/or R⁷ is a group of sub-formula (IV).

Particular examples of groups R⁵, R⁶ and R⁷ are hydrogen, hydroxy, haloor alkoxy. In particular R⁶ and R⁷ are hydrogen. R⁵ may be hydrogen butin addition is suitably a small subsitutent such as hydroxy, halo ormethoxy.

Particular substituents for R¹ include trifluoromethyl, C₁₋₄alkyl, halo,trifluoromethoxy, C₁₋₄alkoxy, C₁₋₄alkanoyl, C₁₋₄alkanoyloxy, nitro,carbamoyl, C₁₋₄alkoxycarbonyl, C₁₋₄alkylsulphanyl, C₁₋₄alkylsulphinyl,C₁₋₄alkylsulphonyl, sulphonamido, carbamoylC₁₋₄alkyl,N—(C₁₋₄alkyl)carbamoylC₁₋₄alkyl, N—(C₁₋₄alkyl)₂carbamoyl-C₁₋₄alkyl,hydroxyC₁₋₄alkyl or C₁₋₄alkoxyC₁₋₄alkyl.

Additionally or alternatively, two such substituents together may form adivalent radical of the formula —O(CH₂)₁₋₄O— attached to adjacent carbonatoms on the R¹ ring.

Preferred substitutents for R¹ are one or more non-polar substituentssuch as halo.

In particular, R¹ is substituted by one or more halo groups, inparticular chlorine. A particular example of an R¹ group is3,4-dichlorophenyl, 3-fluoro-4-chlorophenyl, 3-chloro-4-fluorophenyl or2,3-dichloropyrid-5-yl.

Examples of groups R² include carboxy; cyano; tetrazol-5-yl; SO₃H;—CONHR⁸ where R⁸ is selected from cyano, hydroxy, —SO₂R¹² where R¹² isalkyl such as C₁₋₄alkyl, aryl such as phenyl, heteroaryl ortrifluoromethyl, or R⁸ is a group —(CHR¹⁰)_(r)—COOH where r is aninteger of 1-3 and each R¹⁰ group is independently selected fromhydrogen or alkyl such as C₁₋₄ alkyl; or R² is a group —SO₂NHR⁹ where R⁹is an optionally substituted phenyl or an optionally substituted 5 or 6membered heteroaryl group, or a group COR¹⁴ where R¹⁴ is alkyl such asC₁₋₄alkyl, aryl such as phenyl, heteroaryl or trifluoromethyl, or R² isa group of formula (VI)

where R¹⁰ and R¹¹ are independently selected from hydrogen or alkyl,particularly C₁₋₄ alkyl.

Preferably R² is carboxy or a pharmaceutically acceptable salt or esterthereof.

Suitable groups R³ include hydrogen, fluoro, chloro, bromo, iodo,methyl, cyano, trifluoromethyl, hydroxymethyl, alkoxyalkyl such asC₁₋₄alkoxymethyl, methoxy, benzyloxy, carboxyalkoxy such ascarboxymethoxy, methylsulphanyl, methylsulphinyl, methylsulphonyl orcarboxyC₃₋₆cycloalkyl, —(CHR²²)_(r)—NR²³R²⁴ (where r is 0-2, each R²² isindependently hydrogen or alkyl, in particular C₁₋₄alkyl, R²³ and R²⁴are independently selected from H and C₁₋₄alkyl or R²³ and R²⁴ togetherwith the nitrogen to which they are attached form a 5 or 6 membered ringoptionally containing one further heteroatom selected from O, N, S, S(O)or SO₂. Suitably R²³ and R²⁴ together form a heterocylic ring such asmorpholino or piperazinyl.

Other such groups R³ include optionally substituted aryl groups, such asoptionally substituted phenyl or naphthyl group. Suitable substituentsfor phenyl groups R³ include one or more groups selected from chlorine,fluorine, methyl, trifluoromethyl, trifluoromethoxy, amino, formyl,phenyl, methoxy, phenoxy or phenyl.

R³ may comprise a range of substituents as listed above, in particular,hydrogen or a small substituent group such as C₁₋₄alkyl in particularmethyl, or trifluoromethyl, and is preferably hydrogen.

Suitable optional substitutents for the group R¹⁵, R¹⁶ and R¹⁷ as theyappear in the definition of R⁴, include functional groups ashereinbefore defined, as well as aryl or heterocyclyl groups, either ofwhich may themselves be substituted by one or more functional groups orfurther aryl or heterocyclyl groups.

Particular examples of substituents for groups R¹⁵, R¹⁶ and R¹⁷ includeone or more groups selected from halo such as chloro; hydroxy, cyano;amino, mono- or di-alkylamino; C₁₋₄ alkoxy; carboxy; sulphonamido;CONH₂; alkylamido where the alkyl moiety is optionally substituted forexample with a functional groups such as carboxy; morpholino; pyridyl;pyrimidinyl; phenyl optionally substituted by halo such as chloro,hydroxy, alkoxy such as methoxy, carbamoyl, acyl such as acetyl, orhydroxyalkyl where the alkyl group suitably includes at least two carbonatoms, such as hydroxyethyl. Other examples of subsitutents for phenylgroups R¹⁵ is alkanoylamino group such as methoylamino.

Where R¹⁵, R¹⁶ and/or R¹⁷ is a heterocyclyl group, or where R¹⁶ and R¹⁷together form an optionally substituted heterocyclic ring, these may besubstituted by functional groups such as halo or hydroxy, or by alkylgroups such as methyl or ethyl, or alkenyl or alkynyl groups any ofwhich may be subsituted, for example with hydroxy, as well as withfurther heteroaryl groups such as pyridyl. Particular examples ofheterocyclic groups R¹⁵, R¹⁶ and/or R¹⁷ are optionally substitutedthiophenyl, optionally substituted imidazolyl, optionally subtitutedpyridyl.

Thus thiophenyl groups R¹⁵, R¹⁶ and/or R¹⁷ may comprisepyridyl-thiophenyl, whilst an example of a substituted imidazolyl groupfor R¹⁵, R¹⁶ and/or R¹⁷ is methylimidazolyl and halopyridyl inparticular chloropyridyl is an example of a substituted pyridyl moietyfor these groups.

Particular examples of R¹⁵ include alkyl in particular methyl optionallysubstituted by a functional groups or, in particular, a heterocyclylgroup where the heterocyclyl group may be optionally substituted by afunctional group such as halo or hydroxy or by an alkyl group such asmethyl. Preferably, R¹⁵ is a substituted alkyl group. Where thesubstitutent is a functional group, it is preferably a group of formulaNR¹⁹R²⁰ where R¹⁹ and R²⁰ are as defined above. Thus examples ofsubstituted alkyl groups R¹⁵ include morpholinomethyl or alkyl such asmethyl substituted with a substituted alkyl amino group wherein thesubstitutents include carboxy, alkanoyl, phenyl or alkyl sulphonyl.

Other examples of R¹⁵ are heterocylcyl groups which are optionallysubsitituted for example by alkyl such as methyl, functional groups suchas chloro or heterocycyl groups such as pyridyl.

Particular examples of R¹⁶ and R¹⁷ are alkyl such as methyl.

X is CH₂ or SO₂ and is preferably CH₂.

Suitable pharmaceutically acceptable salts of compounds of formula (I)include acid addition salts such as methanesulfonate, fumarate,hydrochloride, hydrobromide, citrate, maleate and salts formed withphosphoric and sulphuric acid. In another aspect suitable salts are basesalts such as an alkali metal salt for example sodium, an alkaline earthmetal salt for example calcium or magnesium, an organic amine salt forexample triethylamine, morpholine, N-methylpiperidine,N-ethylpiperidine, procaine, dibenzylamine, N,N-dibenzylethylamine oramino acids for example lysine. There may be more than one cation oranion depending on the number of charged functions and the valency ofthe cations or anions. A preferred pharmaceutically acceptable salt is asodium salt.

An in vivo hydrolysable ester of a compound of the formula (I)containing carboxy or hydroxy group is, for example, a pharmaceuticallyacceptable ester which is hydrolysed in the human or animal body toproduce the parent acid or alcohol.

Suitable pharmaceutically acceptable esters for carboxy include alkylesters, such as C₁₋₆ alkyl esters for example, ethyl esters,C₁₋₆alkoxymethyl esters for example methoxymethyl, C₁₋₆alkanoyloxymethylesters for example pivaloyloxymethyl, phthalidyl esters,C₃₋₈cycloalkoxy-carbonyloxyC₁₋₆alkyl esters for example1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters forexample 5-methyl-1,3-dioxolen-2-onylmethyl; andC₁₋₆alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyland may be formed at any carboxy group in the compounds of thisinvention.

Suitable pharmaceutically acceptable esters of compounds of formula (I)are in vivo hydrolysable ester of a compound of the formula (I)containing a hydroxy group includes inorganic esters such as phosphateesters and α-acyloxyalkyl ethers and related compounds which as a resultof the in vivo hydrolysis of the ester breakdown to give the parenthydroxy group. Examples of α-acyloxyalkyl ethers include acetoxymethoxyand 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysableester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyland substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkylcarbonate esters), dialkylcarbamoyl andN-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),dialkylaminoacetyl and carboxyacetyl.

Esters which are not in vivo hydrolysable are useful as intermediates inthe production of the compounds of formula (I) and therefore these forma further aspect of the invention.

Thus examples of compounds of formula (I) include the following: TABLE 1

Compd No. R³ R⁴ R⁵ R⁶ R^(a) R^(b) 1 H

H H H H 2 H

H H Cl Cl 3 H

H H Cl Cl 4 H

H H Cl Cl 5 H

H H Cl Cl 6 H

H H Cl Cl 7 H

H H Cl Cl 8 H NHC(O)CH₂NHCH₂COOH H H Cl Cl 9 H

H H Cl Cl 10 H OC(O)N(CH₃)₂ H H Cl Cl 11 H

H H Cl Cl 12 H

H H Cl Cl 13 H

H H Cl Cl 14 H NHC(O)CH₂N(CH₃)CH₂COOH H H Cl Cl 15 H

H H Cl Clwhere * indicates the point of attachment of the group to the indolering.

Compounds of formula (I) are suitably prepared by methods such as thosedescribed in International Patent Application Nos. PCT/GB98/02340 andPCT/GB98/02341.

In particular compounds of formula (I) where R⁴ is NHCOR¹⁵ or NHSO₂R¹⁵can be prepared by reacting a compound of formula (VII)

where X, R¹, R³, R⁵, R⁶ and R⁷ are as defined in relation to formula(I). R² is a group R² as defined in relation to formula (I) or aprotected form thereof, with a compound of formula (VIII)Z-R²²  (VIII)where Z is a leaving group and R²² is a group COR^(15′) or SO₂R^(15′)where R^(15′) is group R¹⁵ as defined in relation to formula (I) or aprecursor thereof;and thereafter if desired or necessary:

-   (i) converting a precursor group R^(15′) to a group R¹⁵ and/or    converting a group R¹⁵ to a different such group;-   (ii) deprotecting a group R^(2′) to a group R².    -   Suitable leaving groups Z include halo such as chloro.        The reaction is suitably effected in an organic solvent such as        dichloromethane or tetrahydrofuran in the presence of a base        such as triethylamine or pyridine. Moderate temperatures, for        example from 0° to 50° C. and conveniently ambient temperature,        are employed in the reaction.

Compounds of formula (I) where R⁴ is a group OCONR¹⁶R¹⁷ may be preparedby a broadly similar method by reacting a compound of formula (VIIA)

where X, R^(2′), R1, R³, R⁵, R⁶ and R⁷ are as defined in relation toformula (I), R² is a group R² as defined in relation to formula (I) or aprotected form thereof, with a compound of formula (VIIIA)Z-CONR¹⁶R¹⁷  (VIIIA)where Z, R¹⁶ and R¹⁷ are as defined above.

Compounds of formula (VIIA) can be prepared by reacting a compound offormula (IX)

where R³, R⁵, R⁶ and R⁷ are as defined in relation to formula (I) andR^(2′) is as defined in relation to formula (VII) and R⁴⁰ is aprotecting group; with compound of formula (X)R¹—X-Z¹  (X)where R¹ and X are as defined in relation to formula (I) and Z¹ is aleaving group, and thereafter, removing the protecting group R⁴⁰.

Suitable leaving groups for Z include halide such as chloride, bromideor iodide, as well as mesylate or tosylate. The reaction is suitablyeffected in an organic solvent such as dimethylformamide (DMF)tetrahydrofuran (THF) or DCM in the presence of a base such as sodiumhydride, sodium hydroxide, potassium carbonate. Optionally the reactionis effected in the presence of a suitable phase transfer catalyst. Thechoice of base and solvent is interdependent to a certain extent in thatcertain solvents are compatible with some bases only as is understood inthe art. For example, sodium hydride may preferably be used withdimethylformamide or tetrahydrofuran and sodium hydroxide is preferablyused with dichloromethane and a phase transfer catalyst.

The reaction can be carried out at moderate temperatures, for examplefrom 0 to 50° C. and conveniently at about ambient temperature.

Preferably, R^(2′) is an ester group in the compound of formula IX andthis may be subsequently converted to an acid or to another ester orsalt, by conventional methods later in the process. For example, when Xis a group SO₂ and R² is a methyl ester of carboxy, it may be convertedto the corresponding carboxylic acid by reaction with lithium iodide indry pyridine or DMF.

Suitable protecting groups R⁴⁰ include acetyl or benzyl. The reactionconditions employed will be variable depending upon the nature of theprotecting group R⁴⁰ and would be apparent to a skilled person. Acetylgroups may be removed by reaction with a strong base such as sodiummethoxide, whereas benzyl groups may be removed by hydrogenation forexample in the presence of a catalyst such as a palladium catalyst.

Compounds of formula (IX) may be prepared by cyclisation of a compoundof formula (XII)

where R⁵, R⁶, R⁷ and R⁴⁰ are as defined above and R⁴² and R⁴³ representa combination of moieties which can cyclise to form an appropriatelysubstituted pyrrole ring. For example, one of R⁴² and R⁴³ can be a groupof formula —CH═C(R⁴⁴)N₃ where R⁴⁴ is a group R² as defined above, or aprotected form thereof, and the other may be hydrogen. Cyclisation toform a compound of formula (XII) may then be effected by heating forexample under reflux in an organic solvent, in particular a high boilingaprotic solvent such as xylene or toluene.

Alternatively, one of R⁴² and R⁴³ may be nitro and the other may be agroup of formula —CH₂C(O)R² where R^(2′) is as defined above in relationto formula (VII). These compounds will cyclise in the presence of acatalyst such as palladium on carbon in the presence of hydrogen. Thereaction may be effected at moderate temperatures for example of from 0to 80° C., conveniently at about ambient temperature.

Thus examples of compounds of formula (XII) include compounds of formula(XIII) and (XIV)

Compounds of formula (XIII) where R³ is hydrogen may be prepared forexample by reacting a compound of formula (XV)

with a compound of formula (XVI)N₃CH₂R^(2′)  (XVI)where R⁵, R⁶, R⁷, and R^(2′) are as defined hereinbefore. The reactionmay be effected in an organic solvent such as ethanol at lowtemperatures of from −20 to 0° C., suitably at about 0° C. The reactionis suitably effected in the presence of a base such as an alkoxide, inparticular an ethoxide, for example potassium ethoxide.

Compounds of formula (XVI) are suitably prepared by reacting a compoundof formula (XVII)R₄₇CH₂R^(2′)  (XVII)where R^(2′) is defined above and R⁴⁷ is a leaving group such as halideand in particular bromide, with an azide salt, such as an alkali metalazide salt in particular sodium azide.

Compounds of formula (XIV) may be prepared by reacting a compound offormula (XVIII)

where R⁵, R⁶, R⁷, R³, R⁴⁰ and R^(2′) are as defined above, with acompound of formula (XIX)

where R^(2′) is as defined above and R⁴⁸ leaving group such as hydroxy.Examples of compounds of formula (XVI) are oxalates such asdiethyloxalate. The reaction is suitably effected in the presence of abase such as sodium hydride in an organic solvent such as THF. Moderatetemperatures of from 0° to 40° C. and conveniently ambient temperatureis employed.

Compounds of formula (VII) are suitably prepared using a reactionanalogous to that between compounds (IX) and (X), where in place of thecompound of formula (IX), a compound of formula (IXA) is employed

where R^(2′), R³, R⁵, R⁶ and R⁷ are as defined above. Such compounds maybe obtained by reduction of the corresponding nitro compound of formula(XX)

where R^(2′), R³, R⁵, R⁶ and R⁷ are as defined above.

Compounds of formula (X), (XVI), (XV), (XVII), (XVIII), (XIX) and (XX)are either known compounds or they may be prepared from known compoundsby conventional literature methods.

According to a further aspect of the invention there is provided acompound of the formula (I) as defined herein, or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof, for use in amethod of treatment of the human or animal body by therapy. Inparticular, the compounds are used in methods of treatment ofinflammatory disease.

According to a further aspect of the present invention there is provideda method for antagonising an MCP-1 mediated effect in a warm bloodedanimal, such as man, in need of such treatment, which comprisesadministering to said animal an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt, or an in vivohydrolysable ester thereof.

The invention also provides a pharmaceutical composition comprising acompound of formula (I) as defined herein, or a pharmaceuticallyacceptable salt, or an in vivo hydrolysable ester thereof, incombination with a pharmaceutically acceptable diluent or carrier.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, hard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder) or for parenteral administration (for example as a sterileaqueous or oily solution for intravenous, subcutaneous, intramuscular orintramuscular dosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulationinclude, for example, inert diluents such as lactose, sodium carbonate,calcium phosphate or calcium carbonate, granulating and disintegratingagents such as corn starch or algenic acid; binding agents such asstarch; lubricating agents such as magnesium stearate, stearic acid ortalc; preservative agents such as ethyl or propyl p-hydroxybenzoate, andanti-oxidants, such as ascorbic acid. Tablet formulations may beuncoated or coated either to modify their disintegration and thesubsequent absorption of the active ingredient within thegastrointestinal track, or to improve their stability and/or appearance,in either case, using conventional coating agents and procedures wellknown in the art.

Compositions for oral use may be in the form of hard gelatin capsules inwhich the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules in which the active ingredient is mixed with water oran oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxyethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),colouring agents, flavouring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil (such as arachis oil, olive oil, sesame oil orcoconut oil) or in a mineral oil (such as liquid paraffin). The oilysuspensions may also contain a thickening agent such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, or a mineral oil, such as for exampleliquid paraffin or a mixture of any of these. Suitable emulsifyingagents may be, for example, naturally-occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soya bean,lecithin, an esters or partial esters derived from fatty acids andhexitol anhydrides (for example sorbitan monooleate) and condensationproducts of the said partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such asglycerol, propylene glycol, sorbitol, aspartame or sucrose, and may alsocontain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example a solution in 1,3-butanediol.

Suppository formulations may be prepared by mixing the active ingredientwith a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Suitable excipients include, forexample, cocoa butter and polyethylene glycols.

Topical formulations, such as creams, ointments, gels and aqueous oroily solutions or suspensions, may generally be obtained by formulatingan active ingredient with a conventional, topically acceptable, vehicleor diluent using conventional procedure well known in the art.

Compositions for administration by insulation may be in the form of afinely divided powder containing particles of average diameter of, forexample, 30μ or much less, the powder itself comprising either activeingredient alone or diluted with one or more physiologically acceptablecarriers such as lactose. The powder for insufflation is thenconveniently retained in a capsule containing, for example, 1 to 50 mgof active ingredient for use with a turbo-inhaler device, such as isused for insufflation of the known agent sodium cromoglycate.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

For further information on Formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 0.5mg to 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient. For furtherinformation on Routes of Administration and Dosage Regimes the reader isreferred to Chapter 25.3 in Volume 5 of Comprehensive MedicinalChemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press1990.

The size of the dose for therapeutic or prophylactic purposes of acompound of the Formula I will naturally vary according to the natureand severity of the conditions, the age and sex of the animal or patientand the route of administration, according to well known principles ofmedicine. As mentioned above, compounds of the Formula I are useful intreating diseases or medical conditions which are due alone or in partto the effects of farnesylation of rats.

In using a compound of the Formula I for therapeutic or prophylacticpurposes it will generally be administered so that a daily dose in therange, for example, 0.5 mg to 75 mg per kg body weight is received,given if required in divided doses. In general lower doses will beadministered when a parenteral route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 0.5 mg to30 mg per kg body weight will generally be used. Similarly, foradministration by inhalation, a dose in the range, for example, 0.5 mgto 25 mg per kg body weight will be used. Oral administration is howeverpreferred.

A further aspect of the invention comprises the use of a compound offormula (I) as defined above in the preparation of a medicament for thetreatment of inflammatory disease.

The invention is further illustrated, but not limited by the followingExamples in which the following general procedures were used unlessstated otherwise.

Preparation 1

Ethyl N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxylate

Ethyl 4-nitroindole-2-carboxylate (26 g) [prepared according to S. M.Parmerter et. al J. Amer. Chem. Soc., 1958, 80, 4621],3,4-dichlorobenzyl chloride (16 ml), potassium carbonate (17 g) andpotassium iodide (2 g) in DMF (250 ml) were stirred at 60° C. for 2hours. The reaction was concentrated in vacuo and the residuepartitioned between water and dichloromethane. Iso-hexane was added tothe combined organic extracts resulting in crystallisation of theproduct as yellow needles (39 g, 89%) NMR d (CD₃SOCD₃) 1.30 (t, 3H),4.32 (q, 2H), 5.93 (s, 2H), 6.88 (dd, 1H), 7.18 (d, 1H), 7.52 (d, 1H),7.56 (dd, 1H), 7.78 (s, 1H), 8.17 (m, 2H), M/z (+)395 (MH⁺), 393.

Preparation 2

Ethyl N-benzyl-4-aminoindole-2-carboxylate

A mixture of ethyl 4-nitroindole-2-carboxylate (8.2 g), anhydrouspotassium carbonate (6.0 g) and benzyl bromide (4.3 ml) in DMF (100 ml)was stirred at 50-60° C. for 2 hours. The solvent was evaporated invacuo and the residue partitioned between dichloromethane and water (250ml each); the organic layer was separated, dried (MgSO₄) and evaporatedto give a yellow solid (12 g), which was dissolved in a mixture oftetrahydrofuran/ethanol (200 ml, 1:1) and stirred while adding asolution of sodium dithionite (26 g) in water (50 ml). The mixture wasstirred for 1 hour at 25° C. and partitioned between dichloromethane andwater (200 ml each), the organic layer was washed with water (100 ml)and dried (MgSO₄). Combined organic extracts were concentrated in vacuoand the residue purified by column chromatography using dichloromethaneas eluent to give a the product as a brown solid (1.4 g. 14%); NMR d(CD₃SOCD₃) 1.28 (t, 3H), 4.27 (q, 2H), 5.57 (s, 2H), 5.73 (s, 2H), 6.22(d, 1H), 6.62 (d, 1H), 6.95-7.05 (m, 3H), 7.15-7.30 (m, 3H), 7.60 (s,1H).

Preparation 3

Ethyl N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxylate

Sodium hydroxide (3M, 20 ml) was added to a vigorously stirred solutionof ethyl 4-nitroindole-2-carboxylate (4 g), 3,4-dichlorobenzyl chloride(4.73 ml) and tetra-n-butylammonium hydrogensulphate (0.2 g) indichloromethane (60 ml). The reaction was stirred for 48 hours thenpartitioned between 2M HCl and dichloromethane. Combined organicextracts were dried (MgSO₄) and concentrated in vacuo and the residuepurified by column chromatography using iso-hexane: 20% ethyl acetate aseluent to give the product as a yellow crystalline solid (5.26 g, 78%);NMR δ (CD₃SOCD₃) 1.3 (t, 3H), 4.3 (q, 2H), 5.95 (s, 2H), 6.9 (m, 1H),7.6 (m, 4H), 8.2 (t, 2H); M/z (+) 393.3 (M⁺).

Ethyl N-(3,4-dichlorobenzyl)-4-aminoindole-2-carboxylate

A solution of ethyl N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxylate(2.41 g) in tetrahydrofuran (100 ml) was stirred in the presence oftitanium trichloride (1.5% aqueous solution. 50 ml) at room temperatureovernight. The reaction was treated with 40% sodium hydroxide solutionand extracted with 5% methanol in dichloromethane. Combined organicextracts were dried (MgSO₄) and concentrated in vacuo to Live theproduct as a brown solid (1.98 g, 89%); NMR d (CD₃SOCD₃) 1.3 (t, 3H),4.2 (q, 2H), 5.7 (s, 4H), 6.2 (d, 1H), 6.6 (d, 1H), 7.0 (m, 2H), 7.25(m, 1H), 7.5 (d, 1H), 7.6 (m, 1H); M/z (+) 363.3 (MH⁺).

Preparation 4

Ethyl 4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate

Ethyl 4-amino-N-(3,4-dichlorobenzyl)indole-2-carboxylate (2.03 g),chloroacetyl chloride (0.5 ml) and triethylamine (4.0 ml) were stirredin dichloromethane (50 ml) for 16 hours. The reaction was washed withwater, dried (MgSO₄) and concentrated in vacuo. The residue wastriturated with toluene to give the product as a pale grey solid (1.61g, 65%); NMR d (CD₃SOCD₃) 1.28 (t, 3H), 4.30 (q, 2H), 4.40 (s, 2H), 5.81(s, 2H), 6.88 (dd, 1H), 7.30 (m, 3H), 7.50 (d, 1H), 7.76 (s, 1H), 7.78(d, 1H), 10.19 (brs, 1H); M/z (−) 439 (M⁺), 437.

EXAMPLE 1

Compound 2

Ethyl 4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (0.15g) and morpholine (2.0 ml) were dissolved in methoxyethanol (5.0 ml) andthe reaction stirred for 72 hours. The reaction was then poured intowater (100 ml) and the resulting solid filtered and dried in vacuo. Thesolid was dissolved in THF (2.5 ml) and methanol (2.5 ml), and to thiswas added NaOH (3M, 2.0 ml). The reaction was stirred for 16 hours, thenconcentrated. The residue was dissolved in water, and precipitated bydropwise addition of acetic acid. The resulting solid was filtered anddried in vacuo to give the title compound as a white solid (0.1 g, 63%,2 steps); NMR d (CD₃SOCD₃) 2.58 (t, 4H), 3.29 (s, 2H), 3.65 (t, 4H),5.82 (s, 2H), 6.90 (dd, 1H), 7.30 (m, 3H), 7.52 (m, 2H), 7.72 (d, 1H),9.80 (s, 1H); M/z (−) 462 (M⁺), 460, 418.

EXAMPLE 2

The procedure described in Example 1 above was repeated using theappropriate amines. Thus were obtained the compounds described below.

Compound 3

49% yield, 2 steps; NMR d (CD₃SOCD₃) 2.27 (s, 3H), 2.54 (t, 4H), 2.62(t, 4H), 3.22 (s, 2H), 5.84 (s, 2H), 6.95 (dd, 1H), 7.22 (m, 2H), 7.33(s, 1H), 7.41 (s, 1H), 7.50 (d, 1H), 7.72 (d, 1H), 9.75 (s, 1H); M/z (−)475 (M⁺), 473, 429, 109.

Compound 6

14% yield, 2 steps; M/z (−) 510 (M⁻), 508, 464.

EXAMPLE 3

Di-ester of Compound 8

Ethyl 4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (0.4g), glycine methyl ester hydrochloride (0.57 g) and triethylamine (1.25ml) were dissolved in methoxyethanol (4.0 ml) and the reaction heated at100° C. for 6 hours. The reaction was cooled and partitioned betweenwater and ethyl acetate. Combined organic extracts were dried (MgSO₄)and concentrated and the residue purified by chromatography usingtoluene ethyl acetate (1:1) as eluent to give the product, ethyl4-[(N-(methoxycarbonylmethyl)-glycyl)amino]-N-(3,4-dichlorobenzyl)indole-2-carboxylate,as a pale yellow solid (0.17 g, 38%); NMR d (CD₃SOCD₃) 1.28 (t, 3H),3.44 (s, 2H), 3.50 (s, 2H), 3.63 (s, 3H), 4.28 (q, 2H), 5.82 (s, 2H),6.88 (dd, 1H), 7.10-7.30 (m, 4H), 7.50 (d, 1H), 7.69 (s, 1H), 7.80 (dd,1H), 10.00 (brs, 1H); M/z (+) 494, 492 (M⁺).

EXAMPLE 4

Di-ester of Compound 11

Methanesulphonyl chloride (0.1 ml) was added to stirred solution ofethyl4-[(N-(methoxycarbonylmethyl)glycyl)amino]-N-(3,4-dichlorobenzyl)indole-2-carboxylate(0.33 g) and triethylamine (0.47 ml) in dichloromethane (4.0 ml) and thereaction stirred for 3 hours. The reaction was poured into water andextracted with ethyl acetate. Combined organic extracts were dried(MgSO₄) and concentrated and the residue triturated with ether, filteredand dried in vacuo to give the product as a white solid (0.24 g, 63%);NMR d (CD₃SOCD₃) 1.27 (t, 3H), 3.10 (s, 3 H), 3.67 (s, 3H), 4.20 (s,2H), 4.28 (q+s, 2H+2H), 5.82 (s, 2H), 6.87 (dd, 1H), 7.28 (m, 3H), 7.50(d, 1H), 7.80 (m, 2H), 10.00 (brs, 1H); M/z (+) 572, 570 (M⁺).

EXAMPLE 5

The procedure described in the Example 4 above was repeated using theappropriate acid chloride. Thus was obtained the compound describedbelow.

Di-ester of Compound 12

64% yield; M/z (−) 534 (M⁺), 532.

EXAMPLE 6

Di-ester of Compound 14

Sarcosine ethyl ester hydrochloride (1.23 g) and potassium carbonate(1.11 g) were added to a solution of ethyl4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (700 mg) inacetone (25 ml), stirred and heated at 65° C. overnight. The reactionwas partitioned between water (50 ml) and ethyl acetate (50 ml),extracted with ethyl acetate (2×50 ml), and dried (MgSO₄). The combinedorganic extracts were concentrated in vacuo, and the residue purified bycolumn chromatography using 30% ethyl acetate:toluene as eluent, toafford the product as a yellow solid (768 mg, 92%); NMR d (CD₃SOCD₃)1.21 (t, 3H), 1.28 (t, 3H), 2.45 (s, 3H), 3.42 (s, 2H), 3.53 (s, 2H),4.16 (q, 2H), 4.30 (q, 2H), 5.81 (s, 2H), 6.88 (d, 1H), 7.27 (m, 2H),7.52 (d, 1H), 7.67 (s, 1H), 7.84 (d, 1H), 9.95 (s, 1H), M/z (+) 520.3(MH⁺)

EXAMPLE 7

The procedure described in Example 6 above was repeated using theappropriate amine. Thus was obtained the compound described below.

Diester of Compound 13

93% yield; NMR d (CD₃SOCD₃) 1.15 (t, 3H), 1.28 (t, 3H), 3.52 (s, 3H),3.57 (s, 3H), 3.87 (s, 2H), 4.10 (q, 2H), 4.31 (q, 2H),5.83 (s, 2H),6.90 (d, 1H), 7.15-7.44 (m, 8H), 7.53 (d, 1H), 7.67 (s, 1H), 7.83 (d,1H); M/z (+) 596.5 (MH⁺).

EXAMPLE 8

Di-ester of Compound 15

A solution of methyl iodide (0.026 ml) in DMF (2 ml) was added to asolution of sodium hydride (15 mg, 60% in mineral oil) and ethyl4-[(N-benzyl-N-ethoxycarbonylmethyl)glycyl]amino-N-(3,4-dichlorobenzyl)indole-2-carboxylate(the diester of Compound 13) ( 200 mg) in DMF (4 ml), and stirred underan atmosphere of argon at ambient temperature for 4 hours. The reactionwas quenched with water (50 ml) and extracted with ethyl acetate (3×50ml), and the combined organic extracts were dried (MgSO₄), andconcentrated in vacuo to afford the product as a pale brown oil (93 mg,45%); NMR d (CD₃SOCD₃) 1.05 (t, 3H), 1.30 (t, 3H), 3.21 (s, 2H), 3.28(s, 3H), 3.41 (s, 2H), 3.70 (s, 2H), 3.93 (q, 2H), 4.30 (q, 2H), 5.84(s, 2H), 6.90 (d, 1H), 7.01 (d, 1H), 7.07-7.40 (m, 8H), 7.48-7.64 (m,2H); M/z (+) 610.5 (MH⁺).

EXAMPLE 9

Compound 8

Ethyl4-[(N-(methoxycarbonylmethyl)glycyl)amino]-N-(3,4-dichlorobenzyl)indole-2-carboxylate(0.15 g) was dissolved in THF/methanol (1:1) (10 ml) and sodiumhydroxide (2M, 2.5 ml) was added and the reaction stirred for 16 hours.The reaction was then concentrated in vacuo and the residue dissolved inwater. The solution was acidified by dropwise addition of acetic acid,resulting in the precipitation of a white solid which was filtered,washed with water and dried in vacuo to give the desired end product asa white solid (108 mg, 79%); NMR d (CD₃SOCD₃) 3.40 (s, 2H), 3.64 (s,2H), 5.82 (s, 2H), 6.92 (dd, 1H), 7.20-7.38 (m, 3H), 7.50 (d, 1H), 7.62(s, 1H), 7.78 (d, 1H), 10.15 (brs, 1H).

EXAMPLE 10

The procedure described in Example 9 above was repeated using theappropriate ester. Thus were obtained the compounds described below.

Compound 11

79% yield; NMR d (CD₃SOCD₃) 3.10 (s, 3H), 4.02 (s, 2H), 4.20 (s, 2H),5.83 (s, 2H), 6.88 (dd, 1H), 7.25 (m, 3H), 7.50 (d, 1H), 7.75 (s, 1H),7.80 (d, 1H), 10.49 (brs, 1H); M/z (−) 528 (M⁺), 526, 360, 358, 289,253, 217.

Compound 12

78% yield; NMR d (CD₃SOCD₃) 2.00 (d, 3H), 4.03 (s, 1H), 4.20 (s, 1H),4.23 (s, 1H), 4.40 (s, 1H), 5.82 (s, 2H), 6.88 (m, 1H), 7.25 (m, 3H),7.52 (dd, 1H), 7.76 (m, 2H), 10.13 (brs, 1H); M/z (−) 492 (M⁺), 490,324, 253, 224.

Compound 14

60% yield; NMR d (CD₃SOCD₃) 2.46 (s, 31H), 3.38 (s, 2H), 3.42 (s, 2H),5.88 (s, 2H), 6.92 (d, 1H), 7.20 (m, 2H), 7.31 (s, 1H), 7.50 (m, 2H),7.82 (d, 1H), M/z (−) 462.2 (M−H⁺).

Compound 15

15% yield; NMR d (CD₃SOCD₃) 3.21 (s, 2H), 3.31 (s, 3H), 3.40 (s, 2H),3.69 (s, 2H), 5.83 (s, 2H), 6.90 (d, 2H), 6.98 (d, 2H), 7.15 (m, 6H),7.27 (t, 1H), 7.39 (s, 1H), 7.53 (m, 2H); M/z (−) 554.3 (M−H⁺).

Compound 13

25% yield; NMR d (CD₃SOCD₃) 3.44 (s, 2H), 3.46 (s, 2H), 3.85 (s, 2H),5.91 (s, 2H), 6.87 (m, 1H), 7.13-7.36 (m, 6H), 7.40 (m, 2H), 7.53 (m,2H), 7.78 (d, 1H), M/z (−) 538.2 (M−H⁺), 253.2.

EXAMPLE 11

N-Benzyl-4-(2-(pyrid-2-yl)thiophene-5-sulphonyl))aminoindole-2-carboxylicAcid (Compound 1)

To a solution of ethyl N-benzyl-4-aminoindole-2-carboxylate (140 mg) andpyridine (0.08 ml) in dichloromethane (10 ml) at 20° C. was added2-(pyrid-2-yl)thiophene-5-sulphonyl chloride (140 mg) and thc reactionstirred for 2 hours. The mixture was washed with HCl (2M, 10 ml), theorganic layer was concentrated in vacuo and the residue purified bychromatography on silica using ethyl acetate as eluent, to give a yellowsolid which was dissolved in ethanol (50 ml) at 60° C. and treated withNaOH (2M, 4.0 ml) with stirring for 2 hours. The solvent was evaporatedin vacuo, the residue dissolved in water (50 ml) and filtered. The clearyellow filtrate was acidified with 2N HCl and extracted withdichloromethane/methanol (9:1, 100 ml). The organic layer was dried(MgSO₄) and evaporated to give a pale brown solid, which was trituratedwith ether to give the product as an off white powder (150 mg, 63%, 2steps); NMR d (CD₃SOCD₃) 5.87 (s, 2H), 6.9-7.1 (m, 9H), 7.30 (dd, 2H),7.43 (d, 1H), 7.63 (d, 1H), 7.81 (dd, 1H), 7.96 (d, 1H), 8.50 (d, 1H);M/z (−) 488 (M−H⁺).

EXAMPLE 12

The procedure described in Example 11 above was repeated using theappropriate aminoindole and sulphonyl chloride. Thus were obtained thecompounds described below.

4-(4-Acetylaminobenzenesulphonyl)amino-N-(3,4-dichlorobenzyl)indole-2-carboxylicAcid (Compound 4)

66% yield (2 steps); NMR d (CD₃SOCD₃) 2.00 (s, 3H), 5.75 (s, 2H), 6.80(dd, 1H), 6.92 (d, 1H), 7.12 (dd, 1H), 7.22 (m, 2H), 7.48 (d, 1H), 7.56(s, 1H,), 7.66 (s, 4H), 10.24 (brs, 1H), 10.45 (brs, 1H); M/z (−) 532(M−H⁺), 530.

N-(3,4-Dichlorobenzyl)-4-(2-(pyrid-2-yl)thiophene-5-sulphonyl))aminoindole-2-carboxylicAcid (Compound 5)

69% yield (2 steps); NMR d (CD₃SOCD₃) 5.80 (s, 2H), 6.80 (dd, 1H),7.0-7.5 (m, 8H), 7.68 (d, 1H), 7.83 (dd, 1H), 7.92 (d, 1H), 8.48 (dd,1H); M/z (−) 558 (M−H⁺), 556.

N-(3,4-Dichlorobenzyl)-4-(1-methylimidazole-4-sulphonyl)aminoindole-2-carboxylicAcid (Compound 7)

66% yield (2 steps); NMR d (CD₃SOCD₃) 3.60 (s, 3H)) 5.78 (s, 2H), 6.86(dd, 1H),7.04(1H, d), 7.15 (dd, 1H), 7.20 (d, 1H), 7.30 (d, 1H) 7.50 (d,1H), 7.68 (m, 2H), 7.75 (s, 1H), 10.20 (brs, 1H); M/z (−) 479 (M−H⁺),477.

N-(3,4-Dichlorobenzyl)-4-(2-chloropyridyl-5-sulphonyl)aminoindole-2carboxylicAcid

(Compound 9)

30% yield (2 steps); NMR d (CD₃SOCD₃) 5.85 (s, 2H), 6.83 (d, 1H), 6.93(dd, 1H), 7.03 (dd, 1H), 7.15 (d, 1H), 7.20 (s, 1H), 7.26 (s, 1H), 7.46(d, 1H), 7.60 (d, 1H), 8.05 (dd, 1H), 8.62 (d, 1H); M/z(−)512(M−H⁺),510, 508.

EXAMPLE 13 MethylN-(3,4-dichlorobenzyl)-4-(dimethylcarbamyloxy)indole-2-carboxylate(Methyl Ester of Compound 10)

Dimethylcarbamyl chloride (83 mg) was added to a stirred solution ofmethyl N-(3,4-dichlorobenzyl)-4-hydroxyindole-2-carboxylate (150 mg),triethylamine (65 mg),and DMAP (5 mg) in dichloromethane. The reactionwas stirred for 16 hours at room temperature under an atmosphere ofnitrogen. The reaction was washed with hydrochloric acid (2M, 70 ml),saturated aqueous sodium hydrogencarbonate solution, water and saturatedsodium chloride solution. Combined organic extracts were dried (MgSO₄),concentrated in vacuo and the residue purified by column chromatographyusing 60% ethyl acetate:iso-hexane as eluent to give the product as acolourless gum (132 mg, 74%); NMR d (CD₃SOCD₃) 2.94 (s, 3H), 3.12 (s,3H), 3.81 (s, 3H), 5.82 (s, 2H), 6.91 (m, 2H), 7.21 (s, 1H), 7.27-7.36(m, 2H), 7.46 (d, 1H), 7.52 (d, 1H); M/z (+) 421 (MH⁺).

EXAMPLE 14N-(3,4-Dichlorobenzyl)-4-(dimethylcarbamyloxy)indole-2-carboxylic Acid(Compound 10)

Desesterifiation of the compound of Example 13 using the methoddescribed in Example 9 above yielded Compound 10.

93% yield; NMR d (CD₃SOCD₃) 2.94 (s, 3H), 3.11 (s, 3H), 5.91 (s, 2H),6.82 (d, 1H), 6.94-7.03 (m, 2H), 7.18 (t, 1H), 7.29-7.39 (m, 2H), 7.50(d, 1H); M/z (−) 405 (M−H⁺).

EXAMPLE 15 Biological Assays for hMCP-1 Antagonists

The following biological test methods, data and Examples serve toillustrate the present invention.

Abbreviations:

-   ATCC American Type Culture Collection, Rockville, USA.-   BCA Bicinchroninic acid, (used, with copper sulphate, to assay    protein)-   BSA Bovine Serum Albumin-   DMEM Dulbecco's modified Eagle's medium-   EGTA Ethylenebis(oxyethylenenitrilo)tetraacetic acid-   FCS Foetal calf serum-   HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid])-   HBSS Hank's Balanced Salt Solution-   hMCP-1 Human Monocyte Chemoattractant Protein-1-   PBS Phosphate buffered saline-   PCR Polymerase chain reaction

AMPLITAQ™, available from Perkin-Elmer Cetus, is used as the source ofthermostable DNA polymerase.

Binding Buffer is 50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% foetal calfserum, adjusted to pH 7.2 with 1 M NaOH.

Non-Essential Amino Acids (100× concentrate) is: L-Alanine, 890 mg/l;L-Asparagine, 1320 mg/l; L-Aspartic acid, 1330 mg/l; L-Glutamic acid,1470 mg/l; Glycine, 750 mg/l; L-Proline, 1150 mg/l and; L-Serine, 1050mg/l.

Hypoxanthinc and Thymidine Supplement (50× concentrate) is:hypoxanthine, 680 mg/l and; thymidine, 194 mg/l.

Penicillin-Streptomycin is: Penicillin G (sodium salt); 5000 units/ml.Streptomycin sulphate, 5000 μg/ml.

Human monocytic cell line THP-1 cells are available from ATCC, accessionnumber ATCC TIB-202.

Hank's Balanced Salt Solution (HBSS) was obtained from Gibco; see Proc.Soc. Exp. Biol. Med., 1949, 71, 196.

Synthetic cell culture medium, RPMI 1640 was obtained from Gibco; itcontains inorganic salts [Ca(NO₃)₂.4H₂O 100 mg/l; KCl 400 mg/l;MgSO₁.7H₂O 100 mg/l; NaCl 6000 mg/l; NaHCO₃ 2000 mg/l & Na₂HPO₄ (anhyd)800 mg/l], D-Glucose 2000 mg/l, reduced glutathione 1 mg/l, amino acidsand vitamins.

FURA-2/AM is1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraaceticacid pentaacetoxymethyl ester and was obtained from Molecular Probes,Eugene, Oreg., USA.

Blood Sedimentation Buffer contains 8.5 g/l NaCl and 10 g/l hydroxyethylcellulose.

Lysis Buffer is 0.15M NH₄Cl⁻, 10 mM KHCO₃, 1 mM EDTA

Whole Cell Binding Buffer is 50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5%BSA, 0.01% NaN₃, adjusted to pH 7.2 with 1M NaOH.

Wash buffer is 50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% heatinactivated FCS, 0.5M NaCl adjusted to pH7.2 with 1M NaOH.

General molecular biology procedures can be followed from any of themethods described in “Molecular Cloning—A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989).

i) Cloning and Expression of hMCP-1 Receptor

The MCP-1 receptor B (CCR2B) cDNA was cloned by PCR from THP-1 cell RNAusing suitable oligonucleotide primers based on the published MCP-1receptor sequences (Charo et al., 1994, Proc. Natl. Acad. Sci. USA, 91,2752). The resulting PCR products were cloned into vector PCR-II™(InVitrogen, San Diego, Calif.). Error free CCR2B cDNA was subcloned asa Hind III-Not I fragment into the eukaryotic expression vector pCDNA3(InVitrogen) to generate pCDNA3/CC-CKR2A and pCDNA3/CCR2B respectively.

Linearised pCDNA3/CCR2B DNA was transfected into CHO-K1 cells by calciumphosphate precipitation (Wigler et al., 1979, Cell, 16, 777).Transfected cells were selected by the addition of Geneticin Sulphate(G418, Gibco BRL) at 1 mg/ml, 24 hours after the cells had beentransfected. Preparation of RNA and Northern blotting were carried outas described previously (Needham et al, 1995, Prot. Express. Purific.,6, 134). CHO-K1 clone 7 (CHO-CCR2B) was identified as the highest MCP-1receptor B expressor.

ii) Preparation of Membrane Fragments

CHO-CCR2B cells were grown in DMEM supplemented with 10% foetal calfserum. 2 mM glutamine, 1× Non-Essential Amino Acids, 1× Hypoxanthine andThymidine Supplement and Penicillin-Streptomycin (at 50 μgstreptomycin/ml. Gibco BRL). Membrane fragments were prepared using celllysis/differential centrifugation methods as described previously(Siciliano et al., 1990, J. Biol. Chem., 265, 19658). Proteinconcentration was estimated by BCA protein assay (Pierce, Rockford,Ill.) according to the manufacturer's instructions.

iii) Assay

¹²⁵I MCP-1 was prepared using Bolton and Hunter conjugation (Bolton etal., 1973, Biochem. J., 133, 529; Amersham International plc].Equilibrium binding assays were carried out using the method of Ernst etal., 1994, J. Immunol., 152, 3541. Briefly, varying amounts of¹²⁵I-labeled MCP-1 were added to 7 μg of purified CHO-CCR2B cellmembranes in 100 μl of Binding Buffer. After 1 hour incubation at roomtemperature the binding reaction mixtures were filtered and washed 5times through a plate washer (Brandel MLR-96T Cell Harvester) using icecold Binding Buffer. Filter mats (Brandel GF/B) were pre-soaked for 60minutes in 0.3% polyethylenimine prior to use. Following filtrationindividual filters were separated into 3.5 ml tubes (Sarstedt No.55.484) and bound ¹²⁵I-labeled MCP-1 was determined (LKB 1277Gammamaster). Cold competition studies were performed as above using 100pM ¹²⁵I-labeled MCP-1 in the presence of varying concentrations ofunlabelled MCP-1. Non-specific binding was determined by the inclusionof a 200-fold molar excess of unlabelled MCP-1 in the reaction.

Ligand binding studies with membrane fragments prepared from CHO-CCR2Bcells showed that the CCR2B receptor was present at a concentration of0.2 pmoles/mg of membrane protein and bound MCP-1 selectively and withhigh affinity (IC₅₀=110 pM, K_(d)=120 pM). Binding to these membraneswas completely reversible and reached equilibrium after 45 minutes atroom temperature, and there was a linear relationship between MCP-1binding and CHO-CCR2B cell membrane concentration when using MCP-1 atconcentrations between 100 pM and 500 pM.

Test compounds dissolved in DMSO (5 μl) were tested in competition with100 pM labelled MCP-1 over a concentration range (0.01-50 μM) induplicate using eight point dose-response curves and IC₅₀ concentrationswere calculated.

Compounds tested of the present invention had IC₅₀ values of 50 μM orless in the hMCP-1 receptor binding assay described herein. For exampleCompound 2 in Table 1 showed IC₅₀ of 1.17 μM in hMCP-1.

b) MCP-1 Mediated Calcium Flux in THP-1 Cells

The human monocytic cell line THP-1 was grown in a synthetic cellculture medium RPMI 1640 supplemented with 10% foetal calf serum, 6 mMglutamine and Penicillin-Streptomycin (at 50 μg streptomycin/ml, GibcoBRL). THP-1 cells were washed in HBSS (lacking Ca²⁺ and Mg²⁺)+1 mg/mlBSA and resuspended in the same buffer at a density of 3×10⁶ cells/ml.The cells were then loaded with 1 mM FURA-2/AM for 30 min at 37° C.,washed twice in HBSS, and resuspended at 1×10⁶ cells/ml. THP-1 cellsuspension (0.9 ml) was added to a 5 ml disposable cuvette containing amagnetic stirrer bar and 2.1 ml of prewarmed (37° C.) HBSS containing 1mg/ml BSA, 1 mM MgCl₂ and 2 mM CaCl₂. The cuvette was placed in afluorescence spectrophotometer (Perkin Elmer, Norwalk, Conn.) andpreincubated for 4 min at 37° C. with stirring. Fluorescence wasrecorded over 70 sec and cells were stimulated by addition of hMCP-1 tothe cuvette after 10 sec. [Ca²⁺]i was measured by excitation at 340 nmand 380 nm alternately and subsequent measurement of the intensity ofthe fluorescence emission at 510 nm. The ratio of the intensities of theemitted fluorescent light following excitation at 340 nm and 380 nm,(R), was calculated and displayed to give and estimate of cytoplasmic[Ca²⁺] according to the equation:-${\left\lbrack {Ca}^{2 +} \right\rbrack i} = {K_{d}\frac{\left( {R - {R\quad\min}} \right)}{\left( {{R\quad\max} - R} \right)}\left( {{Sf2}/{Sb2}} \right)}$where the K_(d) for FURA-2 Ca²⁺ complex at 37° C. was taken to be 224nm. R_(max) is the maximal fluorescence ratio determined after additionof 10 mM lonomycin, R_(min) is the minimal ratio determined by thesubsequent addition of a Ca²⁺ free solution containing 5 mM EGTA, andSf2/Sb2 is the ratio of fluorescence values at 380 nm excitationdetermined at R_(min) and R_(max), respectively.

Stimulation of THP-1 cells with hMCP-1 induced a rapid, transient risein [Ca ²⁺]_(i) in a specific and dose dependent manner. Dose responsecurves indicated an approximate EC₅₀ of 2 nm. Test compounds dissolvedin DMSO (10 μl) were assayed for inhibition of calcium release by addingthem to the cell suspension 10 sec prior to ligand addition andmeasuring the reduction in the transient rise in [Ca²⁺]i. Test compoundswere also checked for lack of agonist activity by addition in place ofhMCP-1.

c) hMCP-1 and RANTES Mediated Chemotaxis.

In vitro chemotaxis assays were performed using the human monocytic cellline THP-1. Cell migration through polycarbonate membranes was measuredby enumerating those passing through either directly by Coulter countingor indirectly by use of a colourimetric viability assay measuring thecleavage of a tetrazolium salt by the mitochondrial respiratory chain(Scudiero D. A. et al. 1988, Cancer Res., 48, 4827-4833).

Chemoattractants were introduced into a 96-well microtitre plate whichforms the lower well of a chemotaxis chamber fitted with a PVP-free 5 μmporesize polycarbonate adhesive framed filter membrane (NeuroProbe MBseries, Cabin John, Md. 20818, USA) according to the manufacturer'sinstructions. The chemoattractant was diluted as appropriate insynthetic cell culture medium, RPMI 1640 (Gibco) or supplemented with 2mM glutamine and 0.5% BSA, or alternatively with HBSS with Ca²⁻ and Mg²⁺without Phenol Red (Gibco) plus 0.1% BSA. Each dilution was degassedunder vacuum for 30 min and was placed (400 μl) in the lower wells ofthe chamber and THP-1 cells (5×10⁵ in 100 μl RPMI 1640+0.5% BSA) wereincubated in each well of the upper chamber. For the inhibition ofchemotaxis the chemoattractant was kept at a constant submaximalconcentration determined previously (1 nM MCP-1) and added to the lowerwell together with the test compounds dissolved in DMSO (final DMSOconcentration <0.05% v/v) at varying concentrations. The chamber wasincubated for 2 h at 37° C. under 5% CO₂. The medium was removed fromthe upper wells which were then washed out with 200 μl physiologicalsaline before opening the chamber, wiping dry the membrane surface andcentrifuging the 96-well plate at 600 g for 5 min to harvest the cells.Supernatant (150 μl) was aspirated and 10 μl of cell proliferationreagent, WST-1,{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-phenyldisulfonate} plus an electron coupling reagent (Boehringer Mannheim,Cat. no. 1644 807) was added back to the wells. The plate was incubatedat 37° C. for 3 h and the absorbance of the soluble formazan product wasread on a microtitre plate reader at 450 nm. The data was input into aspreadsheet, corrected for any random migration in the absence ofchemoattractant and the average absorbance values, standard error of themean, and significance tests were calculated. hMCP-1 inducedconcentration dependent cell migration with a characteristic biphasicresponse, maximal 0.5-1.0 nm.

In an alternative form of the above assay, fluorescently tagged cellscan be used in order to assist in end point detection. In this case, theTHP-1 cells used are fluorescently tagged by incubation in the presenceof 5 mM Calcein AM (Glycine.N,N′-[[3′,6′-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-diyl]bis(methylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[(acetyloxy)methyl]ester:Molecular Probes) for 45 minutes in the dark. Cells are harvested bycentrifugation and resuspended in HBSS (without Phenol Red) with Ca²⁺,Mg²⁺ and 0.1% BSA. 50 μl (2×105 cells) of the cell suspension are placedon the filter above each well and, as above, the unit is incubated at37° C. for 2 hours under 5% CO₂. At the end of the incubation, cells arewashed off the upper face of the filter with phosphate buffered saline,the filter removed from the plate and the number of cells attracted toeither the underside of the filter or the lower well estimated byreading fluorescence at 485 nm excitation, 538 nm emission wavelengths(fmax, Molecular Devices). The data was input into a spreadsheet,corrected for any random migration in the absence of chemoattractant andthe average fluorescence values, standard error of the mean, percentageinhibition and IC₅₀ of compounds under test and significance tests canbe calculated. In addition to MCP-1 induced chemotaxis, this alternativeform of the assay was also used to measure inhibition of RANTES (2 nM)induced chemotaxis.

d) Binding to Human Peripheral Blood Mononuclear Cells (PBMCs)

i) Preparation of Human PBMCs

Fresh human blood (200 ml) was obtained from volunteer donors, collectedinto sodium citrate anticoagulant to give a final concentration of0.38%. The blood was mixed with Sedimentation Buffer and incubated at37° C. for 20 minutes. The supernatant was collected and centrifuged at1700 rpm for 5 minutes (Sorvall RT6000D). The pellet obtained wasresuspended in 20 ml RPMI/BSA (1 mg/ml) and 4×5 mls of cells werecarefully layered over 4×5 mls of Lymphoprepä (Nycomed) in 15 mlcentrifuge tubes. Tubes were spun at 1700 rpm for 30 minutes (SorvallRT6000D) and the resultant layer of cells was removed and transferred to50 ml Falcon tubes. The cells were washed twice in Lysis Buffer toremove any remaining red blood cells followed by 2 washes in RPMI/BSA.Cells were resuspended in 5 mls of Binding Buffer. Cell number wasmeasured on a Coulter counter and additional binding buffer was added togive a final concentration of 1.25×10⁷ PBMCs /ml.

ii) Assay

[¹²⁵I]MCP-1 was prepared using Bolton and Hunter conjugation (Bolton etal. 1973, Biochem. J., 133, 529; Amersham International plc].Equilibrium binding assays were carried out using the method of Ernst etal., 1994, J. Immunol., 152, 3541. Briefly, 50 μl of ¹²⁵I-labeled MCP-1(final concentration 100 pM) was added to 40 μl (5×10⁵ cells) of cellsuspension in a 96 well plate. Compounds. diluted in Whole Cell BindingBuffer from a stock solution of 10 mM in DMSO were added in a finalvolume of 5 μl to maintain a constant DMSO concentration in the assay of5%. Total binding was determined in the absence of compound.Non-specific binding was defined by the addition of 5 μl cold MCP-1 togive a final assay concentration of 100 nM. Assay wells were made up toa final volume of 100 μl with Whole Cell Binding Buffer and the platessealed. Following incubation at 37° C. for 60 minutes the bindingreaction mixtures were filtered and washed for 10 seconds using ice coldWash Buffer using a plate washer (Brandel MLR-96T Cell Harvester).Filter mats (Brandel GF/B) were pre-soaked for 60 minutes in 0.3%polyethylenimine plus 0.2% BSA prior to use. Following filtrationindividual filters were separated into 3.5 ml tubes (Sarstedt No.55.484) and bound ¹²⁵I-labeled MCP-1 was determined (LKB 1277Gammamaster).

Test compound potency was determined by assay in duplicate using sixpoint dose-response curves and IC₅₀ concentrations were determined.

Compound No. 13 in Table 1 showed 94% inhibition at 20 μm.

No physiologically unacceptable toxicity was observed at the effectivedose for compounds tested of the present invention.

EXAMPLE 16 Pharmaceutical Compositions

The following Example illustrates, but is not intended to limit,pharmaceutical dosage forms of the invention as defined herein (theactive ingredient being termed “Compound X”), for therapeutic orprophylactic use in humans: (a) Tablet I mg/tablet Compound X.  100Lactose Ph. Eur  182.75 Croscarmellose sodium  12.0 Maize starch paste(5% w/v paste)   2.25 Magnesium stearate   3.0 (b) Tablet II mg/tabletCompound X  50 Lactose Ph. Eur  223.75 Croscarmellose sodium   6.0 Maizestarch  15.0 Polyvinylpyrrolidone (5% w/v paste)   2.25 Magnesiumstearate   3.0 (c) Tablet III mg/tablet Compound X   1.0 Lactose Ph. Eur 93.25 Croscarmellose sodium   4.0 Maize starch paste (5% w/v paste)  0.75 Magnesium stearate   1.0 (d) Capsule mg/capsule Compound X  10Lactose Ph. Eur  488.5 Magnesium   1.5 (e) Injection I (50 mg/ml)Compound X  5.0% w/v 1M Sodium hydroxide solution 15.0% v/v 0.1MHydrochloric acid to adjust pH to 7.6 Polyethylene glycol 400  4.5% w/vWater for injection to 100% (f) Injection II (10 mg/ml) Compound X  1.0%w/v Sodium phosphate BP  3.6% w/v 0.1M Sodium hydroxide solution 15.0%v/v Water for injection to 100% (g) Injection III (1 mg/ml. buffered topH6) Compound X  0.1% w/v Sodium phosphate BP 2.26% w/v Citric acid0.38% wlv Polyethylene glycol 400  3.5% w/v Water for injection to 100%(h) Aerosol I mg/ml Compound X  10.0 Sorbitan trioleate  13.5Trichlorofluoromethane  910.0 Dichlorodifluoromethane  490.0 (i) AerosolII mg/ml Compound X   0.2 Sorbitan trioleate   0.27Trichlorofluoromethane  70.0 Dichlorodifluoromethane  280.0Dichlorotetrafluoroethane 1094.0 (j) Aerosol III mg/ml Compound X   2.5Sorbitan trioleate   3.38 Trichlorofluoromethane  67.5Dichlorodifluoromethane 1086.0 Dichlorotetrafluoroethane  191.6 (k)Aerosol IV mg/ml Compound X   2.5 Soya lecithin   2.7Trichlorofluoromethane  67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane  191.6 (l) Ointment ml Compound X  40 mgEthanol 300 μl Water 300 μl 1-Dodecylazacycloheptan-2-one  50 μlPropylene glycol to 1 mlNote:Compound X in the above formulation may comprise a compound illustratedin Examples. The above formulations may be obtained by conventionalprocedures well known in the pharmaceutical art. The tablets (a)-(c) maybe enteric coated by conventional means, for# example to provide a coating of cellulose acetate phthalate. Theaerosol formulations (h)-(k) may be used in conjunction with standard,metered dose aerosol dispensers, and the suspending agents sorbitantrioleate and soya lecithin may be replaced by an alternative #suspending agent such as sorbitan monooleate, sorbitan sesquioleate,polysorbate 80, polyglycerol oleate or oleic acid.

1. A compound of formula (I)

X is CH₂ or SO₂ R¹ is an optionally substituted aryl or heteroaryl ring;R² is carboxy, cyano, —C(O)CH₂OH, —CONHR⁸, —SO₂NHR⁹, tetrazol-5-yl,SO₃H, or a group of formula (VI)

where R⁸ is selected from hydrogen, alkyl, aryl, cyano, hydroxy, —SO₂R¹²where R¹² is alkyl, aryl, heteroaryl, or haloalkyl, or R⁸ is a group—(CHR¹³)_(r)—COOH where r is an integer of 1-3 and each R¹³ group isindependently selected from hydrogen or alkyl; R⁹ is hydrogen, alkyl,optionally substituted aryl such as optionally substituted phenyl oroptionally subtituted heteroaryl such as 5 or 6 membered heteroarylgroups, or a group COR¹⁴ where R¹⁴ is alkyl, aryl, heteroaryl orhaloalkyl; R¹⁰ and R¹¹ are independently selected from hydrogen oralkyl, particularly C₁₋₄alkyl; R³ is hydrogen a functional group,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted alkoxy, optionally substitutedaralkyl, optionally substituted aralkyloxy, optionally substitutedcycloalkyl; R⁴ is a group NHCOR¹⁵, NHSO₂R¹⁵ or OCONR¹⁶R¹⁷ where R¹⁵ isoptionally substituted alkyl, optionally substituted aryl or optionallysubstituted heteroaryl and R¹⁶ and R¹⁷ are independently selected fromhydrogen, optionally substituted alkyl, optionally substituted aryl andoptionally substituted heteroaryl, with the proviso that at least one ofR¹⁶ or R¹⁷ is other than hydrogen, or R¹⁶ and R¹⁷ together with thenitrogen atom to which they are attached form an optionally substitutedheterocyclic ring which optionally contains further heteroatoms; and R⁵,R⁶ and R⁷ are independently selected from hydrogen, a functional groupor an optionally substituted hydrocarbyl groups or optionallysubstituted heterocyclic groups; and further provided that when R⁴ is agroup NHCOR¹⁵, R¹⁵ is substituted alkyl, optionally substituted aryl oroptionally substituted heteroaryl.
 2. A compound according to claim 1wherein a group R¹⁵, R¹⁶ and R¹⁷ as they appear in the definition of R⁴,is substituted by at least one functional group, or an aryl orheterocyclyl groups, either of which may themselves be substituted byone or more functional groups or further aryl or heterocyclyl groups. 3.A compound according to any one of the preceding claims wherein R⁴ is agroup NHCOR¹⁵ or NHSO₂R¹⁵ and R¹⁵ is a substituted alkyl group or anoptionally subsituted hetercyclyl or optionally substituted phenylgroup.
 4. A compound according to claim 3 wherein R¹⁵ is alkylsubstituted by a group of formula NR¹⁹R²⁰ where R¹⁹ and R²⁰ areindependently selected from hydrogen or optionally substitutedhydrocarbyl, or R¹⁹ and R²⁰ together form an optionally substituted ringwhich optionally contains further heteroatoms such as S(O)_(m), oxygenand nitrogen, n is an integer of 1 or 2, m is 1 or
 2. 5. A compoundaccording to any one of the preceding claims where R² is carboxy.
 6. Acompound according to any one of the preceding claims wherein R¹ is3,4-dichlorophenyl, 3-fluoro-4-chlorophenyl, 3-chloro-4-fluorophenyl or2,3-dichloropyrid-5-yl.
 7. A compound according to any one of thepreceding claims where X is CH₂.
 8. A process for preparing a compoundaccording to claim 1 which process comprises either (a) where R⁴ isNHCOR¹⁵or NHSO₂R¹⁵, reacting a compound of formula (VII)

where X, R¹, R³, R⁵, R⁶ and R⁷ are as defined in claim 1, and R² is agroup R² as defined in relation to formula (I) or a protected formthereof, with a compound of formula (VIII)Z-R²²  (VIII) where Z is a leaving group and R²² is a group COR^(15′) orSO₂R^(15′) where R^(15′) is group R¹⁵ as defined in relation to formula(I) or a precursor thereof; or (b) where R⁴ is a group OCONR¹⁶R¹⁷,reacting a compound of formula (VIIA)

where X, R^(2′), R1, R³, R⁵, R⁶ and R⁷ are as defined claim 1 and R² isa group R² as defined in claim 1 or a protected form thereof, with acompound of formula (VIIIA)Z-CONR¹⁶R¹⁷  (VIIIA) where Z, R¹⁶ and R¹⁷ are as defined above; andthereafter if desired or necessary: (i) converting a precursor groupR^(15′) to a group R¹⁵ and/or converting a group R¹⁵ to a different suchgroup; (ii) deprotecting a group R^(2′) to a group R².
 9. Apharmaceutical composition comprising a compound according to any one ofclaims 1 to 7 in combination with a pharmaceutically acceptable carrier.10. A compound according to any one of claims 1 to 7 for use in thepreparation of a medicament for use in the treatment of disease mediatedby monocyte chemoattractant protein-1 or RANTES Regulated uponActivation, Normal T-cell Expressed and Secreted), such as inflammatorydisease.